CN104619848B - Cell transfection method - Google Patents

Abstract

The present invention relates to methods of transfecting cells. In particular, the invention relates to methods of transfecting primordial germ cells in avians, and methods of propagating avians having modified traits.

Description

Cell transfection method

Technical Field

The present invention relates to methods of transfecting cells. In particular, the invention relates to methods of transfecting primordial germ cells in avians, and methods of propagating avians having modified traits.

Background

The development of effective techniques to develop transgenic or genetically modified avians is critical to the agricultural and biopharmaceutical industries, as well as to increase our understanding of avian biology through functional genomics research. In the face of population growth, poultry production will play an important role in ensuring global food safety, and advances in biotechnology today, such as the development of transgenic poultry, will help the industry to meet increasing production needs.

More specifically, the use of transgenic technology to modify traits in poultry that cannot be obtained by traditional breeding, such as disease resistance and sex-determining regulation, is now possible and provides great benefit to the poultry industry. The demand for biopharmaceutical proteins has increased rapidly until recently, cell-based in vitro manufacturing systems have been used that produce new recombinant proteins for the treatment of diseases. The use of transgenic livestock as bioreactors for recombinant protein production is now developing as a major alternative to expensive and labor intensive cell-based systems. The development of chicken transgenic technology has led to the development of eggs that are used as bioreactors for high-level production and purification of biopharmaceutical proteins.

There have also been attempts to introduce a selected foreign gene by cloning the foreign gene into a retroviral vector (e.g., reticuloendotheliosis virus or avian leukemia virus), injecting a recombinant virus into a fertilized egg, allowing the virus to infect a developing embryo (e.g., primordial germ cells) to establish a chimeric gonad or egg cell, and using the resulting recombinant to attempt to introduce the foreign gene into offspring. However, the poultry industry is reluctant to use this technology commercially because viruses (in their native state) are pathogens and even different replication-competent viral vectors can sometimes cause tumors, whereas replication-inactive variants require higher or repeated doses. Even replication-defective viral constructs can pose a risk of recombination with endogenous viral envelopes and thereby becoming replication-competent. In addition, these vectors are currently limited to relatively small (e.g., 2kb or less) sized DNA inserts.

There have also been attempts to inject foreign DNA into immature zygotes which are surgically removed from hens. However, this method requires incubation of the developing embryo in a series of surrogate containers. In addition, it requires specialized spawning colonies (laying flock) and repeated practice to obtain the required surgical and technical skills.

An alternative method involves the injection of genetically modified embryonic cells or Primordial Germ Cells (PGCs) into recipient embryos shortly after spawning. In this method, PCG cultures are generated that retain the ability to differentiate into functional egg cells or sperm that produce cells upon integration into a developing embryo. This type of PCG culture can be genetically modified and then injected into recipient embryos. The recipient embryo will typically be modified by gamma irradiation to weaken endogenous primordial germ cells, thereby giving the injected cells a selective advantage of becoming colonized in the gonadal ridges. The modified cell will then mature and produce a sperm or egg cell capable of transmitting the transgene to at least the next generation. However, this technique is very time consuming because it requires the removal of the PGCs from the donor embryos, and their subsequent culturing and reintroduction into the recipient embryos. In addition, the efficiency of birds containing genetically modified PGCs obtained using this technique is low.

Thus, there remains a need for methods of genetically modifying avian primordial germ cells.

Summary of The Invention

The present inventors have found that direct injection of transfection reagents mixed with DNA into the blood of developing avian embryos results in the introduction of DNA into Primordial Germ Cells (PGCs) and the insertion of the DNA into the genome of the avian.

Accordingly, the present invention provides a method for producing an avian comprising genetically modified germ cells, the method comprising:

(i) injecting a transfection mixture comprising a polynucleotide mixed with a transfection reagent into a blood vessel of an avian embryo, whereby the polynucleotide is inserted into the genome of one or more germ cells in the avian.

In one embodiment, the method further comprises (ii) incubating the embryo at a temperature sufficient for the embryo to develop into a bird.

The transfection mixture is preferably injected into avian embryos at about the stage of 12-17PGC migration. In a preferred embodiment, the transfection mixture is injected into avian embryos at stages 13-14.

Although any suitable transfection reagent may be used in the methods of the present invention, transfection reagents comprising cationic lipids are preferred.

In one embodiment, the transfection reagent comprises a monovalent cationic lipid selected from one or more of the following: DOTMA (N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride), DOTAP (1, 2-bis (oleoyloxy) -3-3- (trimethylammonio) propane), DMRIE (1, 2-ditetradecyloxypropyl 3-dimethyl-hydroxyethylammonium bromide) and DDAB (dimethyldioctadecylammonium bromide).

In another embodiment, the transfection reagent comprises a multivalent cationic lipid selected from one or more of: DOSPA (2, 3-dioleyloxy-N- [2- (spermine carboxamide) ethyl ] -N, N-dimethyl-1-propanaminium trifluoroacetate) and DOSPER (1, 3-dioleoyloxy-2- (6-carboxyspermine) propanamide), TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleylspermine), TMTLS (tetramethyltetralauryl spermine), TMTMTMTMTMTMS (tetramethyltetramyristylspermine) and TMDS (tetramethyldioleylspermine spermine).

In another embodiment, the transfection reagent comprises DOSPA (2, 3-dioleyloxy-N- [2- (spermine carboxamide) ethyl ] -N, N-dimethyl-1-propanaminium trifluoroacetate).

In another embodiment, the transfection reagent further comprises a neutral lipid. The neutral lipid may comprise, for example, DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine), or cholesterol.

In a specific embodiment, the transfection reagent comprises a 3:1(w/w) mixture of DOSPA and DOPE prior to mixing the transfection reagent with the polynucleotide.

Advantageously, the method of the invention is suitable for use in a non-retroviral method of introducing a polynucleotide into the genome of a germ cell. Thus, in one embodiment, the polynucleotide further comprises a nucleotide sequence encoding a transposon or zinc finger nuclease.

In a specific embodiment, the transfection mixture comprises a polynucleotide encoding a transposase. The transposase can be encoded by DNA, such as in a plasmid, or alternatively, the polynucleotide encoding the transposase is RNA.

In a particular embodiment, the transposon is selected from Tol2, mini-Tol2, Sleeping Beauty and PiggyBac.

In another embodiment, the polynucleotide comprises a sequence encoding a zinc finger nuclease.

The germ cell that is genetically modified in the avian may be an embryonic germ cell, preferably the cell is a primordial germ cell.

In one embodiment, the injection mixture is injected into an embryo in an egg shell where the embryo develops.

The polynucleotides in the transfection mixture may be RNA molecules or DNA molecules encoding polypeptides, or DNA molecules encoding RNA comprising a double-stranded region. In a specific embodiment, the polynucleotide encodes an RNA comprising a double-stranded region. The RNA molecule may for example be siRNA, shRNA or RNA decoy (decoy).

In another embodiment, the polynucleotide encodes a polypeptide.

In one embodiment, the RNA molecule or polypeptide reduces replication of the virus in the cell as compared to a cell lacking the RNA molecule or polypeptide.

The methods of the invention can be used to target any viral pathogen of an avian. In one embodiment, the virus is an influenza virus.

The invention further provides an avian comprising genetically modified germ cells, wherein the avian is produced by the method of the invention.

The invention further provides a genetically modified germ cell of an avian of the invention, wherein the germ cell comprises a polynucleotide inserted into the genome.

The invention further provides sperm produced by an avian comprising the genetically modified cell of the invention.

The invention further provides eggs produced by an avian comprising the genetically modified cells of the invention.

The invention further provides a method for genetically modifying germ cells in an avian, the method comprising:

(i) injecting a transfection mixture comprising a polynucleotide mixed with a transfection reagent into a blood vessel of an avian embryo contained in an egg; and

(ii) incubating the embryo at a temperature sufficient to allow the embryo to develop into a bird;

wherein the polynucleotide is inserted into the genome of one or more germ cells in the avian.

In another embodiment, the method comprises one or more features of the invention described herein.

The present invention further provides a method for producing a genetically modified avian, the method comprising:

(i) obtaining an avian comprising a genetically modified germ cell of the invention;

(ii) producing progeny from the avian reproduction comprising genetically modified germ cells; and

(iii) selecting progeny comprising the polynucleotide inserted into the genome.

The invention further provides genetically modified avians produced by the methods of the invention.

The present invention further provides a method of producing a food product, the method comprising:

(i) obtaining an avian comprising the genetically modified germ cell of the invention or a genetically modified avian of the invention; and

(ii) producing a food product from said bird.

In one embodiment, the method comprises harvesting meat and/or eggs from the avian.

The present invention further provides a method of breeding a genetically modified avian, the method comprising:

(i) performing the method of the invention to produce a bird or offspring;

(ii) allowing the bird or progeny to develop into a sexually mature avian; and

(iii) breeding the sexually mature bird to produce a genetically modified bird.

In one embodiment, the present invention provides genetically modified avians produced according to the methods of the present invention.

The present invention further provides a method of modulating a trait in an avian, the method comprising:

(i) injecting a transfection mixture comprising a polynucleotide mixed with a transfection reagent into a blood vessel of an avian embryo, whereby the polynucleotide is inserted into the genome of one or more germ cells in the avian; and

(ii) incubating the embryo at a temperature sufficient to allow the embryo to develop into a bird;

wherein the polynucleotide encodes a polypeptide that modulates an avian trait or an RNA molecule comprising a double stranded region.

In one embodiment, the RNA molecule comprises an siRNA, shRNA or RNA decoy.

In one embodiment, the trait is selected from muscle mass, sex, nutritional composition and/or disease resistance.

The invention further provides a method of increasing resistance of an avian to a virus, the method comprising performing the method of the invention, wherein the polynucleotide is an siRNA, shRNA or RNA decoy that reduces replication of the virus in a cell, or the polynucleotide encodes an antiviral peptide that reduces replication of the virus in a cell.

In a specific embodiment, the virus is an influenza virus.

The invention further provides a bird produced according to the method of the invention.

In certain embodiments of the invention, the avian is selected from a chicken, a duck, a turkey, a goose, a bantam or a quail.

In another embodiment of the methods of the invention, the transfection mixture further comprises a targeting nuclease, or a polynucleotide encoding a targeting nuclease, such that the polynucleotide integrates into the genome of the germ cell. For example, the targeted nuclease may be selected from the group consisting of a zinc finger nuclease, TALEN, and CRISPR.

The invention further provides a method for producing an avian comprising genetically modified germ cells, the method comprising:

(i) injecting a transfection mixture comprising a polynucleotide mixed with a transfection reagent into a blood vessel of an avian embryo, whereby the polynucleotide is inserted into the genome of one or more germ cells in the avian; and

(ii) incubating the embryo at a temperature sufficient for the embryo to develop into a bird;

wherein the transfection reagent comprises a cationic lipid, the polynucleotide further comprises a sequence encoding a transposon, and the transfection mixture is injected into the blood vessels of avian embryos at stages 13-14.

In one embodiment, the transfection reagent comprises a liposome (Lipofectamine)2000 or 3:1(w/w) mixture of DOSPA and DOPE, the transposon is Tol2 or mini-Tol2, and the transfection reagent comprises a polynucleotide encoding a Tol2 transposase prior to mixing with the polynucleotide.

The invention further provides a method for producing an avian comprising genetically modified germ cells, the method comprising:

(i) injecting a transfection mixture comprising a polynucleotide mixed with a transfection reagent into a blood vessel of an avian embryo, whereby the polynucleotide is inserted into the genome of one or more germ cells in the avian; and

(ii) incubating the embryo at a temperature sufficient for the embryo to develop into a bird;

wherein the transfection reagent comprises a cationic lipid and a neutral lipid, the polynucleotide further comprises a sequence encoding a zinc finger nuclease, and the transfection mixture is injected into the blood vessels of avian embryos at stages 13-14.

In one embodiment, the transfection reagent comprises a mixture of DOSPA and DOPE in liposomes of 2000 or 3:1(w/w) prior to mixing the transfection reagent with the polynucleotide.

It will be apparent that preferred features and characteristics of one aspect of the invention may be used in many other aspects of the invention.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The invention is described below by way of the following non-limiting examples and with reference to the following figures.

Drawings

FIG. 1: the EGFP-encoding DNA complexed with liposome 2000 was injected directly into avian embryos. Fluorescence (left) and matched brightfield (right) images of gonads taken from day 7 embryos.

FIG. 2: DNA encoding EGFP complexed with liposome 2000 was injected directly into day 14 avian embryos. Fluorescence (right) and matched brightfield (left) images of gonads taken from day 14 embryos. The last fluorescence image is a close-up of the population of green cells from the left-hand embryo. This region was segmented from the remainder of the gonads stained with chicken vasa homolog (cvh). A small portion of the remaining part of the gonad was used as a negative control.

FIG. 3: the EGFP-encoding DNA complexed with liposome 2000 was injected directly into avian embryos. The PGC marker cvh in the cells was stained. DAPI staining shows nucleic acid material and stains all cells. PGC specific marker cvh stained a subset of cells (brighter gray cells). The arrow indicates transformed cells, which received the directly injected transposon and were stained green.

FIG. 4: in vitro optimization was confirmed by direct injection of avian embryos. EGFP expression in gonads of 14-day embryos.

FIG. 5: the EGFP-encoding DNA complexed with liposome 2000 and a multi-warhead (multi-warhead) construct containing multiple sequences encoding shRNA were injected directly into chicken strain (broiler line) embryos. Fluorescence images from day 12 gonads.

FIG. 6: the EGFP-encoding DNA, multi-warhead construct and extended hairpin construct complexed with liposome 2000 were injected directly into egg laying strain (layer line) chicken embryos. Fluorescence images of embryonic gonads 14 days after direct injection.

FIG. 7: the Tol2-EGFP construct was injected directly with each of two multiple shRNA expression cassettes (pMAT084 and pMAT 085). Images of 10 gonads removed at 14 days, showing EGFP expression.

FIG. 8: gel electrophoresis of the screened PCR products indicated that PB shRNA was integrated into the directly injected embryos. DNA was extracted from PGC-enriched samples from ZFN-treated embryos and from control embryos 5 days after direct injection of ZFN and repair plasmid (containing PB shRNA). Screening PCR was then performed to detect integration of PB shRNA into the genome. Lane 1 shows PB injected embryos, lane 2 control embryos, lane 3 ZFN treated cells (positive control), lane 4 water control.

Key to sequence Listing

1-Tol 2 EGFP construct Polynucleotide sequence

2-Tol 2 transposase amino acid sequence of SEQ ID NO

Oligonucleotide primer of SEQ ID NO 3-Screen 7

4-Screen 6 oligonucleotide primer of SEQ ID NO

5-miniTol 2 Forward oligonucleotide primer of SEQ ID NO

6-miniTol 2 reverse oligonucleotide primer of SEQ ID NO

7-miniTol 2 detection probe of SEQ ID NO

8-genome control region Forward primer of SEQ ID NO

9-genome control region reverse primer of SEQ ID NO

10-genomic control region Probe of SEQ ID NO

Detailed Description

General techniques and definitions

Unless otherwise specifically limited, all technical and scientific terms used herein are to be understood as having the same meaning as commonly understood by one of ordinary skill in the art (e.g., protein chemistry, biochemistry, cell culture, molecular genetics, microbiology and immunology).

Unless otherwise indicated, recombinant DNA and proteins, cell culture and immunological techniques used in the present invention are standard procedures well known to those skilled in the art. The techniques are described and explained throughout the following documents, such as J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J.Sambrook et al, Molecular Cloning A Laboratory Manual,3^rd edn,Cold Spring Harbour Laboratory Press(2001),R.Scopes,Protein Purification–Principals and Practice,3^rdedn, Springer (1994), T.A.Brown (edition), Essential Molecular Biology: A Practical apparatus, Volumes 1 and 2, IRL Press (1991), D.M.Glaver and B.D.Hames (edition), DNA Cloning: A Practical apparatus, Volumes 1-4, IRL Press (1995and 1996), and F.M.Autosubel et al (editors), Current Protocols in Molecular Biology, Grne pub.associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (edition) Antibodies: A Laboratory, Cold mineral, (1988), and John J.J.&Sons (including all updates so far).

The term "avian" as used herein refers to any species, subspecies or breed of organism in the taxonomic "class of avians", such as, but not limited to, the following organisms: chickens, turkeys, ducks, geese, quail, pheasants, parrots, stigmas, hawks, crows, and poultry birds (including ostriches, emus, and turkeys). The term includes breeder chickens (chickens) of different known strains, such as White Leghorn (White Leghorn), Brown Leghorn (Brown Leghorn), rhabdok (Barred-Rock), threx county (susex), New Hampshire (New Hampshire), Rhode Island (Rhode Island), australian black chicken (austrolp), minneak (minoca), athey (amerx), California Gray (California Gray), Italian patience color chicken (Italian partial-chicken), and varieties of turkeys, pheasants, quails, ducks, ostriches, and other commonly commercially raised poultry.

The term "poultry" includes all birds, such as chickens, turkeys, ostriches, pullets (game hen), squabs, guinea fowl, pheasants, quails, ducks, geese and turkeys (emu), which are raised, harvested or domesticated for meat or eggs.

As used herein, "genetically modified avian" or "transgenic avian" refers to any avian wherein one or more cells of the avian contain a heterologous nucleic acid introduced by human intervention.

Direct injection technique

The germ line in chickens is initiated when cells from the ectoderm of the stage X embryo enter the neoendoderm (Kagami et al, 1997; and Petitte, 2002). As endoderm progresses forward, the pre-primordial germ cells are pushed forward into the reproductive crescentia where they can be identified as large glycogen-filled cells. The earliest identification of cells in germ lines by these morphological criteria was about 8 hours after the onset of hatching (stage 4 with a staged system established by Hamburger and Hamilton, (1951)). From stage 4 until it migrates through the vasculature during stages 12-17, primordial germ cells localize to the reproductive crescent. During this time, primordial germ cells are a small population of about 200 cells. As the gonads differentiate, primordial germ cells migrate from the vasculature into the gonadal ridges and integrate into the ovary or testis.

Germ line chimeric chickens have been previously generated by transplanting donor PGCs and gonadal germ cells from different developmental stages (blastoderm to 20-day embryos) into recipient embryos. Methods for obtaining transgenic chickens from long-term cultures of avian Primordial Germ Cells (PGCs) have also been described, for example, in U.S. patent application 20060206952. When combined with a host avian embryo by known procedures, these modified PGCs are transported through germ cell lines to produce genetically modified offspring.

In contrast to the commonly used prior art methods that rely on PGC harvesting from donor embryos, the methods of the present invention involve direct injection of the transfection mixture into avian embryos. Thus, the methods of the invention can be used to transfect avian germ cells including PGCs and embryonic germ cells.

Transfection mixture

In the methods of the invention, the polynucleotide is complexed or mixed with a suitable transfection reagent. The term "transfection reagent" as used herein refers to a composition added to a polynucleotide for enhancing uptake of the polynucleotide by eukaryotic cells including, but not limited to, avian cells such as primordial germ cells. Although any transfection reagent known in the art to be suitable for transfecting eukaryotic cells may be used, the inventors have found that transfection reagents comprising cationic lipids are particularly suitable for use in the methods of the invention. Thus, in a preferred embodiment, the monovalent cationic lipid is selected from one or more of the following: DOTMA (N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride), DOTAP (1, 2-bis (oleoyloxy) -3-3- (trimethylammonio) propane), DMRIE (1, 2-ditetradecyloxypropyl 3-dimethyl-hydroxyethylammonium bromide) or DDAB (dimethyldioctadecylammonium bromide). Preferred multivalent cationic lipids are Lipospermine (Lipospermine), in particular DOSPA (2, 3-dioleyloxy-N- [2- (spermine carboxamide) ethyl ] -N, N-dimethyl-1-propanaminium trifluoroacetate) and DOSPER (1, 3-dioleoyloxy-2- (6-carboxyspermine) propanamide), and di-and tetraalkyltetramethylspermines, including but not limited to TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleylspermine), TMTLS (tetramethyltetralauryl spermine), TMTMTMTMTMS (tetramethyltetramyristylspermine) and TMDS (tetramethyldioleylspermine). The cationic lipids are optionally combined with non-cationic lipids, in particular neutral lipids, such as the following lipids: DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine), or cholesterol. Cationic lipid compositions consisting of a mixture of 3:1(w/w) DOSPA and DOPE or a mixture of DOTMA and DOPE 1:1(w/w) are typically used in the process of the invention. Non-limiting examples of suitable commercially available transfection reagents comprising cationic lipids include liposomes (Life Technologies) and liposome 2000(Life Technologies).

Generally, any dendrimer (dendrimer) useful for introducing nucleic acids into any cell, particularly eukaryotic cells, can be used in the methods of the invention. Dendrimers of generations 5 or higher (G5 or higher) are preferred, with dendrimers of generations G5-G10 being of particular interest. Dendrimers useful in the present invention include dendrimers with NH₃Or dendrimers with ethylenediamine core, GX (NH)₃) Or gx (eda), where X is algebraic. Preferred are dendrimers with X ═ 5 to 10. Dendrimers useful in the present invention include dendrimers in which the repeat units of internal layers are amides (to form polyamides, i.e. PAMAMs). Useful dendrimers include dendrimers in which the terminal functional groups at the outer surface of the dendrimer provide a positive charge density, for example, like terminal amino functional groups. The surface charge and chemical nature of the outer surface of the dendrimer may be varied by altering the functional groups on the surface, for example by reaction of some or all of the surface amino groups. Of particular interest are dendrimers functionalized by reaction with positive amino acids such as lysine or arginine. Grafted dendrimers are described, for example, in PCT applications WO 9622321 and WO9631549 and are mentioned in U.S. Pat. No. 5,266,106, which may be used in the process of the present invention. Activated dendrimers (Haensler and Szoka, 1993; and Tang et al, 1996) can also be used in the methods of the invention.

The transfection reagent may further comprise peptide sequences from viral, bacterial or animal proteins and other sources, including peptides, proteins or fragments or portions thereof that enhance the efficiency of transfection of eukaryotic cells mediated by transfection reagents including cationic lipids and dendrimers. The peptides are described in US20030069173 and include, for example, viral peptides or proteins of influenza virus, adenovirus, Semliki forest virus (Semliki forest virus), HIV, hepatitis, herpes simplex virus, vesicular stomatitis virus or simian virus 40, more specifically, the RGD peptide sequence, NLS peptide sequence and/or VSVG peptide sequence and modified peptides or proteins of any of the foregoing.

The polynucleotide may be mixed (or "complexed") with the transfection reagent according to manufacturer's instructions or known protocols. For example, when plasmid DNA is transfected with Liposome 2000 transfection reagents (Invitrogen, Life Technologies), the DNA can be diluted into 50. mu.l Opit-MEM medium and gently mixed. The liposome 2000 transfection reagents were gently mixed and diluted at appropriate levels into 50. mu.l Opit-MEM medium. After 5 minutes of incubation, the diluted DNA and transfection reagent were combined and gently mixed for 20 minutes at room temperature.

An appropriate volume of transfection mixture may then be injected directly into avian embryos according to the methods of the present invention. Although the appropriate volume may be determined by factors such as the stage of the embryo and the type of avian being injected, the appropriate volume for injecting avian embryos is typically from about 1 μ l to about 3 μ l. One skilled in the art will appreciate that the protocol used to mix the transfection reagent and DNA, as well as the volume to be injected into the avian embryo, can be optimized according to the teachings of the present specification.

Injecting embryos

Prior to injection, the eggs are incubated for about 2.5 days at a temperature suitable for embryo development, e.g., about 37.5 to 38 ℃, with the tip end (tagrion) facing upward (stages 12-17), or until the blood vessels in the embryo are of sufficient size to allow injection. The optimal time for transfection mixture injection is when PGC migration occurs, which typically occurs around stages 12-17, but more preferably stages 13-14. It will be appreciated by those skilled in the art that chickens of the meat line generally have faster growing embryos and thus injection should preferably occur early in stages 13-14 to introduce the transfection mixture into the bloodstream as the PGCs migrate.

Holes are punched in the egg shell in order to access the blood vessels of the avian embryo. For example, a hole of about 10mm may be made in the pointed end of an egg using a suitable tool such as pliers. The shell part and associated membrane were carefully removed while avoiding damage to the embryo and its membrane.

Micropipettes made of siliconized glass capillaries can be used to inject the transfection mixture into the blood vessels of avian embryos. Typically, micropipettes are elongated or "pulled" with a needle puller, with the tip being beveled to a diameter (internal opening) of about 10 μm to about 50 μm, more preferably about 25 μm to about 30 μm, with the aid of a pipette grinder. The micropipette is typically ground to a diameter of about 25 μm to about 30 μm to facilitate injection of the PGC into the avian embryo. It will be appreciated by those skilled in the art that since the transfection mixture is cell-free, narrower diameters may be used in the methods of the invention. The micropipettes produced in this way are also referred to as "drawn glass capillaries".

The drawn glass capillary was loaded with about 1-3. mu.l of transfection complex. Any vessel of sufficient size to accommodate the capillary, such as the limbus or the dorsal aorta, or any other vessel of sufficient size to receive the capillary, may be injected. Air pressure can be used to expel the transfection complex from the capillary into the blood vessel.

Following injection of the transfection mixture into the blood vessels of avian embryos, the eggs are sealed with a sufficient amount of parafilm (parafilm) or other suitable sealing membrane known in the art. For example, if a 10mm hole is made in the shell, the hole can be covered with a paraffin film square of about 20 mm. A warm scalpel blade can then be used to stick the parafilm to the outer surface of the egg. The egg is then inverted to a pointed end down position and incubated at a temperature sufficient to allow the embryo to develop, for example, until subsequent analysis or hatching.

As used herein, the phrases "temperature sufficient for development of the embryo" and "temperature sufficient for development of the embryo into a bird" refer to the incubation temperature required for an avian embryo to develop continuously in an egg, preferably into a bird ready for hatching. Suitable incubation temperatures can be determined by one skilled in the art. For example, eggs are typically incubated at about 35.8 to about 38 ℃. Incubators are commercially available that control the incubation temperature at a desired level, e.g., 37.9 ℃ for 1 to 6 days, about 37.6 ℃ for 9 and 10 days, about 37.5 ℃ for 11 and 12 days, about 37.4 ℃ for 13 days, about 37.3 ℃ for 14 and 15 days, about 37.2 ℃ for 16 days, about 37.1 ℃ for 17 days, and about 35.8 ℃ for 22 days after spawning.

Genomic integration of polynucleotides

To facilitate integration of the polynucleotide into the genome of avian germ cells, transposons, zinc finger nucleases or other non-viral constructs or vectors are preferably used in the methods of the invention.

Examples of suitable transposons include Tol2(Kawakami et al, 2002), mini-Tol2, Sleeping Beauty (Ivics et al, 1997), PiggyBac (Ding et al, 2005), Mariner and Galluhop. The Tol2 transposon was first isolated from Oryzias latipes, a transposon belonging to the hAT family, which is described in Kawakami et al (2000). Mini-Tol2 is a variant of Tol2 and is described in Balciunas et al (2006). When co-acting with the Tol2 transposase, the Tol2 and Mini-Tol2 transposons facilitate integration of the transgene into the genome of the organism. The Tol2 transposase was delivered by a separate non-replicating plasmid, only the Tol2 or Mini-Tol2 transposon and the transgene integrated into the genome, and the plasmid containing the Tol2 transposase was lost in a limited number of cell divisions. Thus, the integrated Tol2 or Mini-Tol2 transposons no longer have the ability to perform subsequent transposition events. In addition, since Tol2 is known not to be a naturally occurring avian transposon, endogenous transposase activity is not present in avian cells, e.g., chicken cells, to cause further transposition events. It is understood in the art that RNA encoding Tol2 transposase may be included in the transfection mixture in place of the DNA plasmid encoding transposase. Thus, Tol2 transposons and transposases are particularly suitable for use in the methods of the invention.

Any other suitable transposon system can be used in the method of the invention. For example, the transposon system may be the Sleeping Beauty, Frog Prince or Mos1 transposon system, or any transposon belonging to the tc1/mariner or hAT family of transposons may be used.

It will be appreciated by those skilled in the art that it may be desirable to include additional genetic elements in the construct to be injected into avian embryos. Examples of additional genetic elements that may be included in the nucleic acid construct include reporter genes, such as one or more genes for fluorescent marker proteins such as GFP or RFP, enzymes that are readily assayed, such as β -galactosidase, luciferase, β -glucuronidase, chloramphenicol acetyltransferase, or secreted embryonic alkaline phosphatase, or proteins with an immunoassay ready therefor, such as hormones or cytokines. Other genetic elements that may be used in embodiments of the invention include genetic elements encoding proteins that confer a selective growth advantage on cells, such as adenosine deaminase, aminoglycoside phosphotransferase, dihydrofolate reductase, hygromycin- β -phosphotransferase, or drug resistance.

Genome editing techniques may also be used in the methods of the invention. For example, the genome editing technology can be a targeted nuclease. The term "targeted nuclease" as used herein includes naturally occurring proteins or modified proteins. In one embodiment, the targeting endonuclease can be a meganuclease (meganuclease). Meganucleases are endodeoxyribonucleases characterized by long recognition sequences, i.e., the recognition sequences are typically about 12 base pairs to about 40 base pairs. As a result of this requirement, the recognition sequence is typically only present once in any given genome. Among meganucleases, the family of homing endonucleases named LAGLIDADG has become a valuable tool for genome and genome editing studies. Meganucleases can be targeted to specific chromosomal sequences by modifying their recognition sequences using techniques well known to those skilled in the art.

In another embodiment, the "targeted nuclease" is a zinc finger nuclease. Zinc Finger Nucleases (ZFNs) are artificial nucleases made by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be modified to target a desired DNA sequence, which allows the zinc finger nuclease to target unique sequences in a complex genome. By utilizing endogenous DNA repair machinery, these agents can be used to precisely alter the genome of higher organisms. Zinc finger nucleases are known in the art and are described, for example, in us patent 7,241,574, reviewed by Durai et al (2005) and Davis and Stokoe (2010).

Prior to the present invention, it was anticipated that zinc finger constructs would be introduced into cultured PGCs in order to modify the PGCs with zinc finger nuclease technology. Transfected cells containing the desired insertion/modification can then be selected and cloned. The sorted and cloned cells will be injected into PGC-depleted recipient embryos.

The inventors have surprisingly found that direct injection of zinc finger nuclease constructs into avian embryos results in specific genome modifications that can be detected in the gonads of 14-day transfected embryos. This finding is surprising, as one would expect the combined level of transfection efficiency and zinc finger nuclease activity to be too low to detect specific modifications in directly injected embryos. In view of the specificity of targeting the desired DNA sequence, and the inventors' discovery that a combination of zinc finger nucleases and transfection reagents injected directly into the embryo achieves higher than expected levels of efficiency, zinc finger nucleases are particularly useful for introducing polynucleotides into the genome of avian germ cells in the methods of the invention.

In another embodiment, the targeting endonuclease can be a transcription activator-like effector (TALE) nuclease (see, e.g., Zhang et al, 2011). TALEs are transcription factors from the plant pathogen xanthomonas that are easily modified to bind new DNA targets. TALEs or truncated forms thereof can be linked to catalytic domains of endonucleases, such as Fokl, to create targeted endonucleases named TALE nucleases or TALENs.

In another embodiment, the "targeted nuclease" is a regularly clustered interspaced short palindromic repeats (CRISPR) nuclease (Barrangou, 2012). CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of specific CRISPR-associated (Cas) genes and non-coding RNA elements capable of programming CRISPR-mediated nucleic acid cleavage. Three classes of (I-III) CRISPR systems have been identified in a wide range of bacterial hosts. One of the main features of each CRISPR locus is the presence of a large number of repeated sequences (direct repeats) separated by short segments of non-repeated sequences (spacers). The non-coding CRISPR array is transcribed and cleaved in direct repeats to form short crrnas containing a single spacer sequence that directs the Cas nuclease to the target site (pro-spacer).

Type II CRISPRs are one of the best known systems (see, e.g., Cong et al, 2013) that perform targeted DNA double strand breaks in four sequential steps. First, two non-coding RNAs, pre-crRNA arrays and tracrrnas are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates the processing of the pre-crRNA into mature crRNA containing a single spacer sequence. Third, the mature crRNA tracrRNA complex directs Cas9 to target DNA through Wastson-Crick base pairing between a spacer on the crRNA and a pre-spacer sequence on the target DNA next to a pre-spacer sequence adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates target DNA cleavage to generate double strand breaks in the pre-spacer sequence. CRISPR systems can also be used to generate single-strand breaks in genomes. The CRISPR system is therefore useful for RNA-guided site-specific genome editing.

Polynucleotide

The methods of the invention can be used to integrate a polynucleotide into the genome of an avian primordial germ cell, which polynucleotide can be delivered to a genetically modified progeny. The polynucleotide integrated into the genome may confer a desired function or activity on a genetically modified cell comprising the polynucleotide, for example, modifying a production trait or increasing disease resistance. Thus, polynucleotides that can be integrated into the genome of a germ cell include polynucleotides encoding short interfering RNAs (sirnas), short hairpin RNAs (shrnas), extended short hairpin RNAs (ehrnas), catalytic RNAs such as ribozymes, RNA decoys, and polynucleotides encoding endogenous or exogenous polypeptides such as polypeptides useful for modulating production traits in avians or increasing disease resistance in avians.

Thus, in some embodiments, the methods of the invention can be used to modify any trait in an avian. Preferred traits that can be modified include production traits and disease resistance. The term "production trait" as used herein refers to any avian phenotype of commercial value, such as muscle mass, sex, disease resistance or nutritional composition. Preferred traits that can be modified according to the methods of the invention include sex, muscle mass and disease resistance. Examples of genes that can be targeted to modify sex as a trait of avian production include DMRT1, WPKCI (ASW), R-spondin, FOX9, aromatase, AMH, and β -catenin.

The term "muscle mass" as used herein refers to the weight of muscle tissue. An increase in muscle mass is determined by weighing the total muscle tissue of birds hatched from eggs treated as described herein, compared to birds from the same species, more preferably the same strain or same breed of avian, even more preferably the same bird (not administered a nucleic acid as defined herein). Alternatively, specific muscles such as the pectoral and/or leg muscles can be used to identify an increase in muscle mass. Genes that can be targeted for use in modulating muscle mass include, for example, the myostatin gene.

RNA interference

In certain embodiments, the methods of the invention utilize nucleic acid molecules encoding double stranded regions for RNA interference, thereby modulating avian traits. The terms "RNA interference", "RNAi" or "gene silencing" generally refer to a method in which a double-stranded RNA molecule reduces the expression of nucleic acid sequences that share substantial or complete homology with the double-stranded RNA molecule. However, RNA interference has been demonstrated to be achieved using non-RNA double stranded molecules (see e.g. US 20070004667).

The double-stranded region should be at least 19 contiguous nucleotides, for example about 19-23 nucleotides, or may be longer, for example 30 or 50 nucleotides, or 100 nucleotides or more. The full length sequence corresponding to the entire gene transcript may be used. Preferably, they are about 19 to about 23 nucleotides in length.

The degree of identity of the double stranded region of the nucleic acid molecule to the targeted transcript should be at least 90%, more preferably 95-100%. The nucleic acid molecule may of course comprise unrelated sequences which may be used to stabilize the molecule.

The term "short interfering RNA" or "siRNA" as used herein refers to a nucleic acid molecule comprising ribonucleotides capable of inhibiting or down-regulating gene expression, for example by mediating RNAi in a sequence-specific manner, wherein the double stranded portion is less than 50 nucleotides in length, preferably about 19 to about 24 nucleotides in length. For example, the siRNA can be a nucleic acid molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or portion thereof. siRNA can be assembled from two separate oligonucleotides, one of which is the sense strand and the other of which is the antisense strand, wherein the antisense and sense strands are themselves complementary.

The term siRNA as used herein is meant to be equivalent to other terms used to describe nucleic acid molecules capable of mediating sequence-specific RNAi, such as micro-rna (mirna), short hairpin rna (shrna), short interfering oligonucleotides (siNA), short interfering modified oligonucleotides, chemically modified siRNA, post-transcriptional gene silencing rna (ptgsrna), and others. In addition, the term RNAi as used herein is intended to be equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational suppression, or epigenetics. For example, the siRNA molecules described herein can be used to epigenetically silence a gene at the post-transcriptional level or at the pre-transcriptional level. In a non-limiting example, the apparent regulation of gene expression by the siRNA molecules described herein can result from siRNA-mediated modification of chromatin structure that alters gene expression.

"shRNA" or "short hairpin RNA" means an RNA molecule in which less than about 50 nucleotides, preferably about 19 to about 23 nucleotides, base pair with a complementary sequence located on the same RNA molecule, and in which the sequence and complementary sequence are separated by an unpaired region of at least about 4 to about 15 nucleotides that forms a single-stranded loop over the stem structure established by the regions of two base complementarity.

The shRNA comprised is a double-or multi-finger hairpin dsRNA in which the RNA molecule comprises two or more such neck loop structures separated by a single-stranded spacer region.

MicroRNA regulation is a specialized branch of the RNA silencing pathway that has evolved towards gene regulation away from traditional RNAi/PTGS. MicroRNAs are a specific class of small RNAs encoded in gene-like elements composed of unique inverted repeats. Upon transcription, microRNA genes produce the neck-loop precursor RNA, which is subsequently processed therefrom. MicroRNAs are typically about 21 nucleotides in length. The released mirnas integrate into RISC-like complexes containing a population of specific Argonaute proteins, displaying sequence-specific gene suppression.

Disease resistance

The methods of the invention can be used to integrate polynucleotides that confer disease resistance to cells into the primordial germ cell genome of avian embryos. For example, a polynucleotide may encode a nucleic acid molecule, such as an siRNA, shRNA, or miRNA, that reduces host or pathogen gene expression, resulting in reduced viral replication in a cell in which the polynucleotide is present. "viral replication" as used herein refers to the amplification of a viral genome in a host cell, packaging of a viral genome in a cell, and/or release of infectious viral particles from a cell.

Alternatively, the polynucleotide may encode an RNA decoy. RNA decoys are known in the art and contain specific nucleotide base sequences that bind to viral proteins essential for the replication of pathogenic viruses. RNA decoys targeting HIV proteins were first described by Sullenger et al (1990). However, it will be appreciated by those skilled in the art that the RNA decoy can be designed to target proteins that play a role in avian viral pathogen replication, such as RNA decoys that target influenza virus polymerase complex proteins.

Preferably, a genetically modified avian comprising a polynucleotide will have increased resistance to a viral pathogen by reducing viral replication in avian cells. Avians herein that are "resistant" or have "increased resistance" to a pathogen or viral pathogen exhibit reduced or no disease symptoms when exposed to the pathogen as compared to susceptible avians. Using the methods of the invention, avians can develop resistance to pathogens such as, but not limited to, influenza virus, marek's disease virus, newcastle disease virus, and infectious bursal disease virus.

In ovo (in ovo) production of recombinant proteins

Petitee and Modziak (2007) described domesticated hens as "very efficient protein bioreactors". It is recognized that avian eggs contain a large amount of protein, and that the majority of proteins, or albumins, are composed of a single species, and there is great potential for the production of recombinant or heterologous proteins in eggs. The difficulties faced by prior art methods of producing transgenic poultry for the production of therapeutic proteins in eggs are well known in the art. Despite the use of undesirable lentiviral systems, the production of transgenic avians has been achieved that store high levels of commercially relevant proteins in eggs. Thus, the methods of the invention can be used to produce genetically modified avians that express heterologous or recombinant polypeptides in ovo. Commercially important proteins that can be produced in eggs include therapeutic proteins such as antibodies and vaccine antigens.

Production and propagation of genetically modified avians

The methods of the invention include methods of breeding genetically modified avians and methods of producing food products from genetically modified avians. It will be appreciated by those skilled in the art that the avian of the invention comprising genetically modified germ cells may be germ line chimeric in that only some of the germ cells that migrate into the gonads are genetically modified. Thus, avians containing genetically modified germ cells can be bred to produce offspring in which all cells are genetically modified. Thus in one embodiment, the present invention provides a method for producing a genetically modified avian, the method comprising: (i) obtaining an avian comprising germ cells genetically modified according to the invention; (ii) producing progeny from the avian reproduction comprising genetically modified germ cells; and (iii) selecting progeny comprising the polynucleotide inserted into the genome.

The avian species comprising the genetically modified germ cells of the invention, as well as the genetically modified avian species according to the invention, are useful in food production. Thus, the process of the present invention can be used to produce poultry products for human and animal consumption. Methods of producing food products from poultry are well known in the art and may comprise harvesting meat and/or eggs from poultry, such as, but not limited to, chickens. In certain embodiments, the avian is genetically modified to include a polynucleotide that modulates a production trait.

Examples

Example 1: direct injection of EGFP expression constructs into embryos

Mu.g of the nucleic acid construct encoding Enhanced GFP (EGFP) (flanked by the Tol2 sequence) and 1.0. mu.g of the plasmid encoding Tol2 transposase were complexed with 3. mu.l of liposome 2000. The complexing of nucleic acids and transfection reagents was performed in a total volume of 90. mu.l of OptiMEM or OptiPRO medium using the manufacturer's (Life Technologies) recommended incubation times.

After a final 20 minute incubation, 1-3. mu.l of the complex was injected into the blood vessels of 2.5 day old chick embryos (stages 13-17, Hamburger and Hamilton, 1951). Blood removal is not required. The embryo is accessed by removing a small portion (10mm) of the shell. After injection, the holes were sealed with a 20mm square parafilm.

Different levels of EGFP expression were observed in most gonads, 7 days and 14 days. Cells isolated from gonads and green cells were also confirmed to be PGCs (fig. 1-3).

Example 2: in vitro optimization of DNA to transfection reagent ratio

Testing of DNA by experiment: optimal proportions of liposomes 2000, and the volume of medium constituting the transfection complex. DNA constructs encoding EGFP and Single hairpin (shRNA) flanked by Tol2 sequences were complexed with liposomes 2000 in OptiMEM in volumes of 50, 40, 30 or 20. mu.l. The ratio of DNA (μ g) used to liposomes 2000(μ l) was as follows: 1:2, 2:4 and 4: 8.

The complexes were transfected into chicken fibroblasts (DF-1) and analyzed for EGFP expression. The results show (not shown) DNA (μ g) in 1: 2: the liposome 2000 ratio, 30. mu.l medium ratio, 50. mu.l medium ratio, was slightly more effective.

The in vitro data was subsequently validated in embryos. Mu.g of DNA construct containing Tol2 transposon, 0.66. mu.g transposase plasmid and 2. mu.l liposome 2000 were complexed in OptiMEM and injected directly into the chick embryos. All surviving embryos had good EGFP expression levels at day 14 (fig. 4).

Example 3: testing FuGene transfection reagent

FuGene (Promega) was tested as a transfection reagent using a similar DNA: Fugene ratio as recommended by the manufacturer for cell culture transfection. The DNA construct complexed with FuGene contained the EGFP expression cassette flanked by the Tol2 sequence. The complex (0.66. mu.g EGFP-Tol2 construct, 1.33. mu.g transposase plasmid, 6. mu.l FuGene) was injected directly into 15 embryos. One of the embryos showed very little expression of EGFP in the 14-day gonad. This experiment was repeated, and at 12 days, all 10 embryos injected were still alive. There were several green cells in the gonads of both embryos.

Example 4: direct injection converted meat lines

Since the previous direct injection experiments were performed on egg strain chickens, the purpose of this experiment was to test whether the direct injection method could be used to successfully transform meat strain chickens. EGFP expression constructs containing single hairpin and flanking Tol2 sequences were complexed with liposomes 2000 (0.33. mu.g transposon construct, 0.66. mu.g transposase, 2. mu.l liposomes 2000) and injected directly into the dorsal aorta of chicken embryos. 12 of the 13 injected embryos survived 10 days and a significant amount of EGFP expression was detected in most gonads.

The experiment was repeated with EGFP expression constructs (0.33. mu.g transposon, 0.66. mu.g transposase, 2. mu.l liposome 2000) containing multiple hairpins (shRNA). A significant amount of EGFP expression was found in day 12 embryos.

Example 5: comparison of OptiMEM and OptiPRO media as transfection reagents

Comparison of OptiMEM (with animal products), OptiPRO (without animal products) and PBSA as transfection reagent media. EGFP expression constructs containing flanking Tol2 sequences were complexed with transfection reagents (0.33. mu.g transposon, 0.66. mu.g transposase, 2. mu.l liposome 2000) and injected directly into chicken embryos. All embryos showed some greenness in the gonads at day 12, and the media used did not affect mortality. OptiMEM and OptiPRO produced comparable results, while PBSA resulted in a significant reduction in EGFP expression in the gonads.

Example 6 injection of egg-laying Strain of chickens with multiple warhead constructs

Both DNA constructs were complexed with transfection reagents and injected directly into the chick embryos. The first DNA construct comprised an EGFP expression cassette and multiple shRNA hairpins flanked by Tol2 sequences, and the second construct comprised an EGFP expression construct and a single stretch hairpin cassette encoding three double-stranded regions. The constructs were complexed with transfection reagents in the following amounts: 0.33. mu.g transposon, 0.66. mu.g transposase, 2. mu.L liposome 2000. EGFP expression was found in most embryonic gonads at day 14 for both constructs.

Example 7: test for Tol2-EGFP durability

The DNA construct containing the EGFP expression cassette, the multiple hairpin and flanked by Tol2 was complexed with transfection reagents (0.33. mu.g transposon, 2. mu.L liposome 2000). The transfection complex without transposase was injected directly into the chick embryos.

Embryos lacking transposase still displayed green cells in some embryos, but less than when transposase was included. This indicates that the plasmid was able to remain in the gonadal cells for at least 2 weeks after direct injection, and not all of the observed green color was due to integration of Tol2 into the genome.

Example 8: animal-free liposomes

The EGFP expression cassette with Tol2 and multiple shRNA expression cassettes was complexed with transfection reagents (Liposome 2000CD) without animal products (0.33. mu.g transposon, 0.66. mu.g transposase, 2. mu.L Liposome 2000 CD). At day 14, all 10 embryonic gonads tested had high EGFP expression (fig. 7).

Example 9: direct injection at 3.5 days

In all previous experiments, transfection complex injections were performed at 2.5 days. The purpose of this experiment was to test embryos injected directly at other times (3.5 days). The DNA construct comprising the EGFP expression construct and Tol2 was complexed with liposome 2000CD (0.33. mu.g transposon, 0.66. mu.g transposase, 2. mu.l liposome 2000 CD).

At day 14, 8 of 21 embryos had low EGFP expression in the gonads. Thus, the timing of the direct injection at day 2.5 is important, and no effective PGC transfection was observed by day 3.5.

Example 10: altering the ratio of transposon to transposase

While maintaining the ratio of DNA to liposome 2000CD to medium, we increased the ratio of transposons in the DNA mixture while slightly decreasing the ratio of transposase plasmids. Since future experiments require more eggs to be injected, we use slightly different volumes. The inventors also tested the removal of blood from the embryo prior to injection of the transfection mixture to determine if this allowed the injection of an increased volume of mixture.

The DNA construct containing the EGFP expression cassette and Tol2 was complexed with transfection reagents (0.66. mu.g transposon, 1.0. mu.g transposase, 3. mu.l liposome 2000 CD). At day 14, the pre-bled embryos had similar levels of EGFP expression in the gonads compared to non-pre-bled embryos. The new DNA ratios worked well and good EGFP expression levels were observed.

Example 11: JetPEI transfection reagent

For JetPEI, the DNA construct containing the EGFP expression cassette and Tol2 was complexed with the transfection reagent (4. mu.g transposon, 6. mu.g transposase, 1.6. mu.l JetPEI (Polyplus transfection)) in 50. mu.l OptiPRO (containing 5% glucose). Upon transfection JetPEI caused blood clotting, but this did not affect embryo viability. Green cells were found in these embryos and gonads, but most of them differed from the transformed PGC phenotype seen when transfected with liposome 2000.

A second experiment was performed to test the JetPEI transfection reagent. Two reaction mixtures were used: i) 0.66. mu.g transposon, 1.0. mu.g transposase, 0.5. mu.l JetPEI in 100. mu.l OptiPRO (containing 5% glucose); and ii) 1.32. mu.g transposon, 2.0. mu.g transposase, 0.5. mu.l JetPEI in 100. mu.l OptiPRO (containing 5% glucose).

Upon transfection JetPEI caused blood clotting and reaction mixture (ii) resulted in improved embryo viability. Some EGFP expression was again found in the gonads, but again the cell types were not PGC-like. Gonads were removed, cells were isolated and stained for PGC markers. None of the green cells showed staining for PGC markers, indicating that PGCs were not transfected by JetPEI complex.

Example 12: zinc finger nucleases

The purpose of this experiment was to determine whether zinc finger nuclease plasmids could be used to transform PGCs by direct injection techniques. The DNA used in this experiment contained two zinc finger nuclease plasmids and overlapping fragments complexed with transfection reagent 0.5. mu.g of each plasmid, 3. mu.l of liposome 2000CD in 90. mu.l of OptiPRO.

Since EGFP was not present on the plasmid, the inventors relied on a PCR test that will amplify the fragment only when the overlapping fragment has integrated into the chicken genome. After 14 days of incubation, the gonads were removed, PGCs were enriched by antibody sorting method, and genomic DNA was prepared. PCR indicated that the overlapping fragments had integrated into the chicken genome. These results demonstrate that zinc finger nucleases are suitable for integrating DNA into the avian PGC genome using the direct injection method of the present invention.

Example 13: results

Following the protocol outlined above, the inventors observed significant transformation of PGCs in recipient embryos and to a greater extent than the methods described in the prior art for transfecting PGCs. By staining cells with markers specific for PGCs, the inventors confirmed that most of the transformed cells in the gonads were PGCs. The inventors cultured the recipient embryos to sexual maturity and were able to detect the Tol2 transposon sequence in > 90% of adult male semen.

Other transfection reagents were used, however lipid-based reagents resulted in better transfection of PGCs. JetPEI did transfect cells by this method, but it could not be confirmed that any of the transfected cells were PGCs. FuGene transfects cells at a very low rate.

Example 14: modification of genomes using direct injection of zinc finger nucleases

A Zinc Finger Nuclease (ZFN) targeting the region of intron 5 of the PANK1 gene was injected into embryos with a plasmid containing anti-influenza shRNA PB1-2257 and the region required for homologous repair, and the embryos were subsequently analyzed for shRNA integration.

A total of 1.5. mu.g of DNA (500. mu.g of each ZFN plasmid, and 500. mu.g of repair plasmid) was added to 45. mu.l of OptiPRO prior to injection into 30 eggs for 2.5 days, and then complexed with 3. mu.l of liposome 2000CD in 45. mu.l of OptiPRO. Eggs were incubated until day 7, at which time the gonads were removed and isolated and MACS sorted with SSEA-1 antibody (Santa Cruz Biotech) to enrich for PGCs. DNA was isolated from PGC enriched samples from ZFN treated and control embryos using Qiagen DNAeasy kit.

Successful integration of the shRNA was screened using PCR with primers that bind to the genome outside the region for homologous repair (Screen 7: 5 'GTGACTCAGACTCCTGTTAG (SEQ ID NO:3)) and primers that bind to the shRNA (Screen 6: 5' TCTGCTGCTTCACAGTCTTC (SEQ ID NO: 4)). PCR was performed using a green master mix (Promega) according to the manufacturer's instructions, cycling conditions were: 2 minutes at 94 ℃ followed by 45 seconds at 94 ℃ and 36 cycles at 55 ℃ for 45 seconds and 1 minute at 72 ℃ for 10 seconds. Followed by a final extension at 72 ℃ for 10 minutes.

PCR was performed on DNA from PGC-enriched samples of ZFN-treated and control embryos, DNA from positive control cells that had previously been demonstrated to have shRNA integration, and water controls. FIG. 8 shows gel electrophoresis of these PCR reactions. The first lane, which contains PCR from ZFN direct injection embryos, clearly shows a band representing genomic integration in the injected embryos.

Example 15: results of direct injection of modifications into the Chicken genome

After several rounds of direct injection, a total of 277 cocks and their semen raised to sexual maturity were tested for the presence of the Tol2 transgene. Of the 277 samples tested, 98 were found to contain the Tol2 transgene with different percent levels of positive semen. Some of these positive G (0) cocks were mated and a total of 7393G (1) chicks were screened. 65 chicks were found to be transgenic. Subsequent matings with these G (1) chicks confirmed the inheritance of the transgene to Mendelian in the G (2) generation.

Hatched chicks were cultured to sexual maturity and the presence of miniTol-EGFP in the semen was detected by quantitative real-time PCR (qPCR). Semen samples were collected and DNA was extracted from 20. mu.l semen diluted in 180. mu.l PBS using Qiagen DNeasy blood and tissue kit according to the manufacturer's instructions. Then the semen genome DNA is diluted in ddH according to 1/100₂O, used for PCR reaction. According to the manufacturer's instructions

qPCR was performed on ep realplex (Eppendorf Hamburg, Germany). Briefly, a 20. mu.l reaction was set up, containing 10. mu.l Taqman 2 × Universal master Mix (Applied Biosystems), 1. mu.l 20 × FAM tapeled Assay Mix (Applied Biosystems) and 9. mu.l diluted DNA. Each sample was set up in duplicate and contained specific primers and probes for minTol2Needle:

a forward primer: 5 'CAGTCAAAAAGTACTTATTTTTTGGAGATCACT 3' (SEQ ID NO: 5);

reverse primer: 5 'GGGCATCAGCGCAATTCAATT 3' (SEQ ID NO: 6);

detecting a probe: 5 'ATAGCAAGGGAAAATAG 3' (SEQ ID NO: 7);

and specific primers and probes to genomic control regions from chicken genome as template controls:

a forward primer: 5 'GATGGGAAAACCCTGAACCTC 3' (SEQ ID NO: 8);

reverse primer: 5 'CAACCTGCTAGAGAAGATGAGAAGAG 3' (SEQ ID NO: 9);

detecting a probe: 5 'CTGCACTGAATGGAC 3' (SEQ ID NO: 10).

The PCR cycle parameters were: the initial annealing step was 95 ℃ for 10 minutes, followed by 95 ℃ for 15 seconds, cycling 45 times, then 60 ℃ for 1 minute. Testing each cock at least twice, if the obtained minTol 2C_TValues below 36 were scored as positive. C of control genomic region_TValues below 32 are used to indicate that the sample tested contains sufficient DNA.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications discussed and/or cited herein are incorporated herein in their entirety.

The present application claims priority to US 61/636,331 filed on day 4/20 of 2012, US 61/783,823 filed on day 14 of 2013 and AU 2013204327 filed on day 12 of 2013, both of which are incorporated herein by reference in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the relevant art in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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Claims (28)

1. A method of producing an avian comprising genetically modified germ cells, the method comprising:

(i) injecting a transfection mixture into a blood vessel of an avian embryo, the transfection mixture comprising:

(a) one or more polynucleotides, wherein at least one of the one or more polynucleotides is to be inserted into the genome of one or more germ cells in the avian embryo;

(b) a transfection reagent comprising a cationic lipid; and

(c) a transposase, a targeted nuclease, or a polynucleotide encoding a transposase or a targeted nuclease, and whereby the at least one polynucleotide is inserted into the genome of one or more germ cells in the avian embryo, and

(ii) incubating the avian embryo at a temperature sufficient for the avian embryo to develop into a bird.

2. The method of claim 1, wherein the transfection mixture is injected into avian embryos at stages 13-14.

3. The method of claim 1, wherein the transfection reagent comprises a monovalent cationic lipid selected from one or more of the following: DOTMA (N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride), DOTAP (1, 2-bis (oleoyloxy) -3-3- (trimethylammonio) propane), DMRIE (1, 2-ditetradecyloxypropyl 3-dimethyl-hydroxyethylammonium bromide) and DDAB (dimethyldioctadecylammonium bromide).

4. The method of claim 1, wherein the transfection reagent comprises a multivalent cationic lipid selected from one or more of: DOSPA (2, 3-dioleyloxy-N- [2- (spermine carboxamide) ethyl ] -N, N-dimethyl-1-propanaminium trifluoroacetate) and DOSPER (1, 3-dioleoyloxy-2- (6-carboxyspermine) propanamide), TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleylspermine), TMTLS (tetramethyltetralauryl spermine), TMTMTMTMTMTMS (tetramethyltetramyristylspermine) and TMDS (tetramethyldioleylspermine spermine).

5. The method of claim 4, wherein the transfection reagent comprises DOSPA (2, 3-dioleyloxy-N- [2- (spermine carboxamide) ethyl ] -N, N-dimethyl-1-propanaminium trifluoroacetate).

6. The method of claim 1, wherein the transfection reagent further comprises a neutral lipid.

7. The method of claim 6, wherein the neutral lipid comprises DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine), or cholesterol.

8. The method of claim 7, wherein the transfection reagent comprises a 3:1w/w mixture of DOSPA and DOPE prior to mixing the transfection reagent with the polynucleotide.

9. The method of claim 1, wherein the polynucleotide inserted into the genome comprises a nucleotide sequence encoding a transposon.

10. The method of claim 9, wherein the transfection mixture comprises a transposase or a polynucleotide encoding a transposase.

11. The method of claim 10, wherein the polynucleotide encoding the transposase is RNA.

12. The method of claim 9, wherein said transposon is selected from the group consisting of Tol2, mini-Tol2, Sleeping Beauty and PiggyBac.

13. The method of claim 1, wherein the targeted nuclease is a zinc finger targeted nuclease, a TALEN targeted nuclease, or a CRISPR targeted nuclease.

14. The method of claim 1, wherein the germ cells are primordial germ cells.

15. The method of claim 1, wherein the injection mixture is injected into an embryo in an egg shell in which the embryo develops.

16. The method of claim 1, wherein the polynucleotide inserted into the genome encodes an RNA molecule comprising a double-stranded region.

17. The method of claim 16, wherein the encoded RNA molecule is an siRNA, shRNA, or RNA decoy.

18. The method of claim 1, wherein the polynucleotide inserted into the genome encodes a polypeptide.

19. The method of claim 16, wherein said RNA molecule reduces viral replication in a cell as compared to a cell lacking said RNA molecule.

20. The method of claim 19, wherein the virus is an influenza virus.

21. The method of claim 18, wherein the polypeptide reduces viral replication in the cell compared to a cell lacking the polypeptide.

22. The method of claim 21, wherein the virus is an influenza virus.

23. Sperm produced by an avian comprising genetically modified germ cells, wherein the avian is produced by the method of any one of claims 1-22.

24. A method for genetically modifying a germ cell of an avian, the method comprising:

(i) injecting a transfection mixture into a blood vessel of an avian embryo, the transfection mixture comprising:

(a) one or more polynucleotides, wherein at least one of the one or more polynucleotides is to be inserted into the genome of one or more germ cells in the avian embryo;

(b) a transfection reagent comprising a cationic lipid; and

(c) a transposase, a targeted nuclease, or a polynucleotide encoding a transposase or a targeted nuclease, and whereby the at least one polynucleotide is inserted into the genome of one or more germ cells in the avian embryo; and

(ii) incubating the avian embryo at a temperature sufficient to allow the avian embryo to develop into a bird.

25. The method of claim 1, further comprising

(i) Propagating the avian comprising the genetically modified germ cell to produce progeny; and

(ii) selecting for progeny of the genetically modified avian comprising the polynucleotide inserted into the genome.

26. The method of claim 25, further comprising producing a food product from the genetically modified avian.

27. The method of claim 26, wherein the method comprises harvesting meat and/or eggs from the genetically modified avian.

28. The method of any one of claims 1-22 and 24-27, further comprising;

(i) allowing the bird or progeny comprising the genetically modified germ cell to develop into a sexually mature avian comprising the genetically modified germ cell; and

(ii) producing genetically modified avian progeny from the sexually mature avian breeding of said germ cell comprising the genetic modification.

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